| According to the updated "List of Microorganisms for Use in Food" issued by the National Health Commission of China in 2022,only the L.cremoris subsp.cremoris and L.lactis subsp.lactis are listed under the genus Lactococcus for use in food.Enterprises must strictly adhere to this list when selecting microorganisms,thus accurate taxonomic status and good growth performance are the primary criteria for microorganism selection by enterprises.In previous studies on the diversity of lactic acid bacteria,our laboratory has accumulated some strains of L.cremoris and L.lactis.At that time,were only identified by 16 S r RNA gene sequencing,so their taxonomic status was not accurate enough.The purpose of this study is to determine the taxonomic status of these strains more accurately(at the subspecies level)and to preliminarily evaluate their growth performance.The 32 strains of Lactococcus were isolated from traditional fermented yoghurt in Tibet and the got of honeybee in Zhejiang.They were identified based on sequence analyses of 16 S r RNA gene and 8 housekeeping genes(phe S,rec A,atp A,rpo A,rpo B,gyr B,hsp60,tuf)as well as whole-genome sequencing.The results showed that 28 strains(YX-2,1255-2,X0854,X0811-2,X0777-2,X0752,ST-1,GYR-2,CQ-1,1375-1,1319,1313,1292,1266,1201,1037,1021,1014-2,991,971-2,F201-1,F-203-1,F-205-1,F-211-1,F-216-4,935-1,206,and 928)were most closely related to L.lactis,while 4 strains(5-2,1030,1245,and X2047)were most closely related to L.cremoris.Representative strains 1319 and 5-2 were selected for comparison with the reference strains:(1)Sequence analysis of 16 S r RNA gene and eight housekeeping genes showed that 1319 had the highest similarity with type strains of L.lactis subsp.hordniae and L.lactis subsp.lactis;5-2 showed the highest similarity with the type strains of L.cremoris subsp.cremoris and L.cremoris subsp.tructae.(2)ANI(average nucleotide sequence identity)between 1319 and the type strains of two subspecies of L.lactis were 97.6% and 98.7%,respectively.The ANI values between 5-2 and the type strains of two subspecies of L.cremoris were 97.8% and 97.9%,respectively which were greater than the critical value of 95.0%-96.0%.(3)d DDH(digital DNA-DNA hybridization)values between 1319 and the type strains of two subspecies of L.lactis were 80.4% and 89.2%,respectively.The d DDH values of 5-2 and the type strains of two subspecies of L.cremoris subspecies were 81.0% and 81.4%,respectively,which were higher than the interspecific critical value of 70.0%.In conclusion,the 28 strains represented by 1319 belonged to L.lactis.Four strains,represented by 5-2,belonged to L.cremoris.Inter-species sequence similarity analyse of the genus Lactococcus indicated that some species that cannot be distinguished based on 16 S r RNA gene can be effectively distinguished using phe S,hsp60,and gyr B genes.phe S,hsp60,and gyr B genes cannot distinguish subspecies.API 50 CH sugar fermentation test showed that 28 strains of L.lactis were most similar to L.lactis subsp.lactis CGMCC 1.1936 T,and they could utilize D-ribose,D-galactose,amygdalin,maltose,cellobiose,and lactose,therefore theses 28 strains could be identified as L.lactis subsp.lactis.Four strains of L.cremoris could utilize D-xylose and cellobiose,but not utilize D-mannitol,melibiose,sucrose,raffinose,and gluconate.API ZYM enzyme activity assay results showed that these four strains could produce cystine arylamidase but not α-galactosidase,α-glucosidase,and β-glucosidase.Strains 5-2,1030,1245,and X2047 showed significant differences from the type strains of two subspecies of L.cremoris,and therefore may be a potential new subspecies of L.cremoris.All 32 strains showed good growth in m MRS liquid medium,entering the stationary or decline phase after 24 hours with a viable cell count of 109 CFU/m L.Two representative strains1375-1(slow growth)and 1021(fast growth),were selected from 28 strains of L.lactis,and two representative strains,1030(slow growth)and 1245(fast growth),were selected from four strains of L.cremoris.These four representative strains were inoculated into fresh cow’s milk and showed slow growth,entering the stationary or decline phase after 48 hours with a viable cell count of 107 CFU/m L for 1030,while the other three strains showed a viable cell count of109 CFU/m L.This study has enriched the strain resource library of L.lactis and L.cremoris,meeting the practical needs of enterprises for these bacteria.It also identified housekeeping genes that can effectively differentiate between species of the Lactococcus genus,providing a theoretical basis for molecular identification at the species level.Additionally,the discovery of a potential new subspecies expands our understanding of the diversity within L.cremoris. |
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